Giant phagocytes (Gφ) and neutrophil-macrophage hybrids in human carotid atherosclerotic plaques - An activated phenotype.

Introduction: A small subpopulation of CD66b+ neutrophils with extended lifespan and immensely large size was identified in vitro. They internalized dead neutrophil remnants and cellular debris, transforming them into giant phagocytes (Gφ) resembling macrophage-foam cells with a massive lipid content and CD68+ scavenger receptor expression. Thus, we sought to investigate if similar CD66b+ neutrophils with altered morphology and functions exist in inflammatory/atherosclerotic conditions in vivo, by using human carotid atherosclerotic plaques. Methods: Thirty-three plaques were obtained from 31 patients undergoing endarterectomy. Carotid plaques were analyzed for CD markers by immunohistochemistry and immunofluorescence and quantitatively analyzed by confocal microscopy. Intra-plaque lipids were stained with Oil Red O. Results: Plaque CD66b+ neutrophils co-expressed myeloperoxidase (MPO)+ and neutrophil elastase (NE)+. Also, co-expression of CD66b+/CD68+, CD66b+/CD36+, CD66b+/vascular-endothelial-growth- factor (VEGF)+ and 3-nitrotyrosine (3-NT)+/NE+ was noted. Similarly, macrophages co-expressed CD163+/CD68+, CD163+/VEGF+ and CD163+/3-NT+. Both cell types were predominantly localized in lipid-rich areas and stained for lipids. CD66b+ and CD163+ expressions were highly positively correlated with each other and each with CD68+, and 3-NT+. Morphologically, CD66+ cells were big, had a rounded nucleus, and resembled macrophage-foam cell morphology as well as that of Gφ in vitro. To clarify whether CD66b+ and CD163+ cells represent two distinct plaque-populations, plaques were double-stained for CD66b/CD163 co-localization. A third of the plaques was negative for CD66b/CD163 co-localization. Other plaques had a low co-localization, but in few plaques, co-localization was high, collectively, indicating that in some of plaques there were two distinct cell populations, those resembling Gφ, and those co-expressing CD66b+/CD163+, demonstrating a hybrid neutrophil-macrophage phenotype. Interestingly, CD66b+/CD163+ co-localization was highly positively correlated with the oxidant 3-NT, hence, supporting trans-differentiation of CD66b+ cells to CD163+ Cells. Conversely, phagocytosis of dead neutrophils by macrophages might have also occurred. Discussion: Thus, we conclude that in some of the plaques CD66b+ cells might represent cells resembling Gφ that developed in prolonged culture conditions. Yet, CD66b+/CD163+ co-expressing cells represent a new neutrophil-macrophage hybrid population of unknown transitioning point, possibly by adopting macrophage markers or contrariwise. Nonetheless, the significance and functions of these cells in plaque biology or other inflammatory/atherosclerotic conditions should be unveiled.

Markers of Carotid Plaque Destabilization in Patients With Sleep-Disordered Breathing.

Sleep-disordered breathing (SDB) is a nightly respiratory condition characterized by intermittent hypoxia, leading to oxidative stress, inflammation, and atherosclerosis. However, most cellular markers of human carotid plaques in SDB have not yet been assessed. We aimed at characterizing the cellular, inflammatory, and nitro-oxidative stress markers in carotid plaques obtained from 25 patients undergoing endarterectomy and screened for SDB. Sleep studies were performed during their preoperative hospitalization night using the Watch-PAT 100 device. Oxygen desaturation index (ODI) was used for dividing patients into two groups. Fourteen patients with ODI >5 were designated as SDB and 11 patients with ODI ≤ 5 as non-SDB. Demographics, comorbidities, cardiovascular risk factors, and medications were recorded. Cellular markers in plaques were analyzed by immunofluorescence using confocal microscopy. The expression of neutrophils was identified by CD66b+ and neutrophil elastase, macrophage-foam cells were identified by CD163+, and scavenger receptors by CD68+ and CD36+ expression. Additional markers included 3-nitrotyrosine, endothelial CD31, and smooth muscle cell-actin (SMC-actin). Plaques' lipids were determined by immunohistochemistry with Oil Red O staining. Notably, significantly higher values were found for SDB as compared to patients with non-SDB for 3-nitrotyrosine (p <0.004) and intracellular lipids' content (p <0.02), whereas SMC-actin was lower (p <0.006). There were no significant differences between patients with carotid-associated symptoms (symptomatic) and patients without carotid-associated symptoms (asymptomatic). However, a sub-group of symptomatic patients with co-existent SDB expressed the highest 3-nitrotyrosin, and intracellular lipids levels, and the lowest SMC-actin levels, whereas non-SDB/asymptomatic patients expressed the lowest 3-nitrotyrosin and lipids levels and the highest SMS-actin levels among all patients. Accordingly, ODI was lowest in non-SDB/asymptomatic patients and highest in SDB/symptomatic. In conclusion, plaques of patients with SDB were characterized by markedly increased levels of 3-nitrotyrosine and intracellular lipids content. Conversely, SMC-actin levels were significantly lower. These three markers, such as increased 3-nitrotyrosine and intracellular lipids and decreased SMC-actin are associated with plaque vulnerability and instability. These findings are in line with earlier reports demonstrating increased intima-media thickness in large cohorts of sleep apnea and patients with SDB, and thus, may indicate a higher susceptibility to plaque vulnerability and rapture in patients with SDB.